Potential antipsoriatic avarol derivatives as antioxidants and inhibitors of PGE2 generation and proliferation in the HaCaT cell line

Article abstract of DOI:10.1021/np049873n

The synthesis and structure-activity relationships for a series of 14 new avarol derivatives as antioxidants and inhibitors of cell proliferation and PGE₂ generation in human keratinocytes are described. Compound 6 (thiosalicylic derivative) was the most potent inhibitor of superoxide generation in human neutrophils and also potently inhibited PGE₂ generation in the human keratinocyte HaCaT cell line. Compound 7 (3′-methylaminoavarone) presented the best antiproliferative profile, by the inhibition of ³H-thymidine incorporation in HaCaT cells, with potency similar to the reference compound anthralin. None of the avarol derivatives showed any sign of cytotoxicity measured as LDH release in treated keratinocytes. The potency and pharmacological profile of derivatives are also discussed.

Full text of DOI:10.1021/np049873n

J . Nat. Prod. 2004, 67, 1459-1463
1459
P oten tia l An tip sor ia tic Ava r ol Der iva tives a s An tioxid a n ts a n d In h ibitor s of
P GE2 Gen er a tion a n d P r olifer a tion in th e Ha Ca T Cell Lin e
Maria Amigo´,† Maria Carmen Terencio,† Maya Mitova,‡,§ Carmine Iodice,‡ Miguel Paya´,† and
Salvatore De Rosa*,‡
Departament de Farmacologia, Facultat de Farma`cia, Universitat de Vale`ncia, Av. V. Andre´s Estelle´s s/ n, 46100, Burjassot,
Valencia, Spain, Istituto di Chimica Biomolecolare CNR, Via Campi Flegrei, 34, 80078 Pozzuoli, Napoli, Italy, and Institute of
Organic Chemistry with Center of Phytochemistry, BAN, 1113 Sofia, Bulgaria
Received April 7, 2004
The synthesis and structure-activity relationships for a series of 14 new avarol derivatives as antioxidants
and inhibitors of cell proliferation and PGE2 generation in human keratinocytes are described. Compound
6 (thiosalicylic derivative) was the most potent inhibitor of superoxide generation in human neutrophils
and also potently inhibited PGE2 generation in the human keratinocyte HaCaT cell line. Compound 7
3
(3′-methylaminoavarone) presented the best antiproliferative profile, by the inhibition of H-thymidine
incorporation in HaCaT cells, with potency similar to the reference compound anthralin. None of the
avarol derivatives showed any sign of cytotoxicity measured as LDH release in treated keratinocytes.
The potency and pharmacological profile of derivatives are also discussed.
Avarol (1) is a marine sesquiterpenoid hydroquinone
possessing a rearranged drimane skeleton with interesting
pharmacological properties including anti-inflammatory,
antitumor, and antiviral actions.1-3 Previous studies dem-
onstrated the antioxidant properties of avarol, which
inhibits superoxide generation and microsomal lipid per-
oxidation.4,5 The biological activities of this compound have
been correlated with its redox chemistry and its ability to
effect radical production, while the terpenoid moiety plays
a marginal role in biological processes.6 These interesting
properties and the previous findings that avarone, the
quinone of avarol, reacts toward protein sulfhydryl groups,7
and that 5′-monoacetyl and diacetyl avarol, from D. avara,8
and some amino derivatives of avarone9,10 also show
interesting biological properties prompted us to prepare
acyl, sulfide, and further amino derivatives of avarol and
evaluate their biological properties.
Psoriasis is a chronic inflammatory skin disease mainly
characterized by abnormal keratinocyte proliferation and
accumulation of polymorphonuclear leukocytes in the
skin.11 Although the pathogenesis of psoriasis still remains
unclear, there is growing evidence supporting the impor-
tance of reactive oxygen species (ROS) in the development
of psoriatic lesions.12,13 Uncontrolled production of active
oxygen species leads to peroxidative damage to membranes
of the skin, a tissue that is particularly vulnerable to the
effects of this species.14 In fact, increased reactive oxygen
species and insufficient antioxidant activity have been
determined in psoriatic lesions.12
limited by the side-effects of irritation and staining of the
uninvolved skin.18,19 The mechanism of anthralin-induced
skin irritation is not completely understood, but several
studies have suggested that it may be associated in part
with the formation of active oxygen species or anthralin-
derived radicals and the subsequent lipid peroxidation.20-23
In the last years, the development of topically active
compounds, which should obviate this drawback, has been
highly desirable for the treatment of psoriatic lesions.24
On the basis of antioxidant properties of avarol and the
role of ROS generation in the pathogenesis of psoriasis, we
undertook the synthesis and possible structure-activity
relationships for 14 avarol derivatives as potential anti-
psoriatic agents by inhibition of superoxide generation in
activated human neutrophils or reduction of cell prolifera-
tion and PGE2 generation in the cultured human kera-
tinocyte HaCaT cell line. In addition, all the compounds
have been tested on keratinocyte cell viability to discard
any sign of cytotoxicity that could explain cell growth
inhibition.
Resu lts a n d Discu ssion
Avarol (1) was isolated from the sponge Dysidea avara,25
collected in the Bay of Naples, Italy. Ester derivatives (3-
5) of avarol were obtained by adding the corresponding acyl
chloride to a solution of avarol in pyridine. The compound
6 was obtained by adding thiosalicylic acid to a solution of
avarone (2), obtained by Ag2O oxidation of avarol, in
ethanol. Amino derivatives (7-16) were generally obtained
by slowly adding the corresponding amine dissolved in
basic solution to a dilute solution of avarone in ethanol or
ethanol-water (1:1).9
For alltheamines (methylamine,vanillylamine,tryptamine,
benzylamine, and p-methoxybenzylamine) two isomers
were obtained with substitution at 3′ (7, 9, 11, 13, and 15)
and 4′ (8, 10, 12, 14, and 16) of the benzoquinone ring, as
previously described9 for other amino derivatives. The
position of the substituent was determined by the analysis
of 1H NMR spectra. Signals of protons in the benzoquinone
ring are doublets in 3′-substituted compounds and singlets
in 4′-substituted compounds.
Eicosanoids have also been suspected to play an impor-
tant role in the pathophysiology of psoriasis. These me-
tabolites affect blood vessels and inflammatory cells,
contributing to dilate capillaries in the dermis and increase
leukocyte infiltration and epidermal cell growth.15 In this
way, keratinocytes are able to enhance the rate of cell
proliferation through increased PGE2.16,17
Anthralin is among the most widely used drugs in the
treatment of psoriasis. However, its clinical efficacy is
* Corresponding author. Tel: +39-0818675029. Fax: +39-0818041770.
E-mail: sderosa@icmib.na.cnr.it.
† Universitat de Vale`ncia.
Initial radical-scavenging properties were obtained using
2,2-diphenyl-1-picrylhydrazyl (DPPH)26,27 as a TLC spray
‡ Istituto di Chimica Biomolecolare CNR.
§ Institute of Organic Chemistry with Center of Phytochemistry.
10.1021/np049873n CCC: $27.50
© 2004 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/16/2004

1460 J ournal of Natural Products, 2004, Vol. 67, No. 9
Sch em e 1. Synthesis and Structure of Compounds 1-16
Amigo´ et al.
reagent. Active compounds were further tested in solution.
Avarol (1) showed the most potent antioxidant activity,
with IC50 18 µM, while 6, 9, and 10 exhibited moderate
potencies, with IC50 34, 95, and 98 µM, respectively.
In the assay of ROS generation in stimulated human
neutrophils, all the compounds were tested at 5 µM. As
expected, avarol (1) (IC50 3.6 µM) and the reference
compound fraxetin inhibited the generation of oxygen-
derived species in a concentration-dependent manner.
Compound 6 also potently reduced this parameter (IC50 2.5
µM), while compounds 7 and 8 exerted only a weak
inhibitory effect at 5 µM. Anthralin did not affect chemi-
luminescence at the concentration used. It has been
reported that during the redox reaction, avarol/avarone
undergoes a one-electron transfer with formation of a
semiquinone free radical that acts as a radical scavenger.5
The potent antioxidant properties showed by compound 6
could also be related to the presence of a hydroquinone
moiety. We believe that the stability of acyl derivatives in
the biological medium with the difficulty to produce free
hydroquinone by hydrolysis is the main aspect of the loss
of their activity in the tested bioassays. The stability of
the quinone form of amino derivatives is at least in part
responsible for the diminution of some aspect of biological
activities, because they can be reduced in biological me-
dium only with difficulty. On the other hand, it should be
noted that none of the avarol derivatives caused cytotox-
icity in the lactate dehydrogenase (LDH) assay.28
The human HaCaT keratinocyte cell line was used as a
model for a highly proliferative epidermis useful in the
evaluation of antipsoriatic agents.29 All the compounds
were tested for antiproliferative activity in comparison to
the antipsoriatic drug anthralin. As shown in Table 1,
compounds 7, 8, 10-12, and 14 significantly reduced cell
proliferation at 5 µM (Table 1). Compound 7 was the most
potent, with an IC50 close to that of anthralin. Results
showed that there were no obvious requirements for the
more potent antiproliferative activity of compound 7 be-
cause only minor structural changes, such as variation of
the position of the methylamino function in the quinone
ring (compound 8), decreased potency. To confirm that
inhibition of keratinocyte growth was not a result of
membrane damage, cell viability was assessed on the basis
of leakage of LDH into the culture medium.28 Treatment
of HaCaT cells with standard anthralin caused remarkable
damage to plasma membrane integrity (59% toxicity at 5
µM) in contrast to compound 7 and the rest of the avarol
derivatives, which do not present any sign of cytotoxicity
(Table 1).
As shown in Table 1, some avarol derivatives also
significantly inhibited the generation of PGE2, compound
6 being the most potent. The pronounced effect of com-
pound 6 on PGE2 levels could be related to the presence of
a thiosalicylic function at the hydroquinone moiety, which
could act through cyclo-oxygenase (COX) inhibition, in a
manner similar to nonsteroidal anti-inflammatory drugs
(NSAIDs).30 As expected, NS-398, used as COX-2 inhibitor
reference compound, potently inhibited PGE2 levels in the
culture cell.
Results obtained in the present study show that it is
difficult to establish a clear relation between inhibition of
keratinocyte cell growth and reduction of superoxide or
PGE2 generation. Although compound 6 was a potent
inhibitor of ROS and PGE2 production, it was not active
as an inhibitor of HaCaT proliferation at 5 µM. In contrast,
compound 7 was the most potent antiproliferative deriva-
tive but presented weak or negative effects on the other
measured parameters. These results suggest a different
mode of action for the compounds and also prove how
difficult it is to find a possible structure-activity relation-
ships. However, compounds 6 and 7 offer interesting
perspectives because their profile and potency may have
relevance in the inhibition of inflammatory response
(compound 6) and psoriatic pathologies (compound 7). It
is interesting to note that compound 7 retains the potent
antiproliferative activity of the antipsoriatic anthralin but
in contrast does not present any cell toxicity. Moreover,
compound 7, by exerting antioxidant properties, could also
contribute to reduce the possible undesirable effects derived

Potential Antipsoriatic Avarol Derivatives
J ournal of Natural Products, 2004, Vol. 67, No. 9 1461
Ta ble 1. Superoxide Generation in Human Neutrophils: Cell Proliferation, PGE2 Production, and Cytotoxicity in the HaCaT
Keratinocyte Cell Line
c
chemiluminescenca
3H-thymidineb
%I (5 µM)
PGE2
LDH released
(A550nm mU)
% I (5 µM)
IC50
IC50
%I (5 µM)
IC50
avarol
3
4
5
6
7
8
9
10
11
12
13
14
15
16
71.7 ( 4.7f
14.2 ( 4.7
11.5 ( 2.2
25.8 ( 5.6
90.2 ( 4.4f
33.8 ( 2.7e
37.8 ( 2.8e
25.5 ( 2.9
36.8 ( 5.2
25.2 ( 3.6
26.7 ( 4.0
26.8 ( 1.1
17.6 ( 2.0
17.3 ( 1.9
10.8 ( 1.7
31.2 ( 6.5
76.4 ( 3.3f
ND
3.6 µM
18.9 ( 2.4
19.6 ( 7.0
12.5 ( 4.1
36.3 ( 5.5
22.6 ( 2.2
73.2 ( 2.9f
33.5 ( 3.9e
16.6 ( 6.9
35.0 ( 3.4e
33.3 ( 3.3e
36.7 ( 4.6
25.6 ( 4.9
40.8 ( 3.8f
24.5 ( 3.6
16.8 ( 4.8
73.8 ( 3.2f
ND
4.9 ( 3.3
39.4 ( 8.5
8.3 ( 6.2
1.6 ( 1.6
151 ( 5
152 ( 1
155 ( 2
158 ( 1
159 ( 2
169 ( 9
157 ( 3
153 ( 1
148 ( 2
151 ( 1
150 ( 2
163 ( 3
158 ( 1
178 ( 1
152 ( 1
285 ( 34f
ND
2.5 µM
72.9 ( 6.5f
14.9 ( 9.7
54.8 ( 3.8f
45.9 ( 9.1
38.9 ( 4.2
47.1 ( 5.3e
50.4 ( 4.4e
47.8 ( 5.1e
32.2 ( 5.1
55.4 ( 4.0f
13.1 ( 8.0
ND
2.6 µM
4.5 µM
anthralin
fraxetin
NS-398
triton X
2.9 µM
1.0 µM
ND
ND
ND
93.8 ( 5.3f
ND
10 nM
ND
ND
376 ( 4f
a
Results show percentages of inhibition (%I) at 5 µM. IC50 was determined for those compounds that reached 50% inhibition. Statistical
evaluation included one-way analysis of variance followed by Dunnett’s t-test for multiple comparisons. ND ) not determined. Superoxide
generation was measured by chemiluminescence with luminol. Fraxetin was used as antioxidant reference compound. Results show
b
percentages of inhibition (%I) at 5 µM. IC50 was determined for those compounds that reached 50% inhibition. Statistical evaluation
included one-way analysis of variance followed by Dunnett’s t-test for multiple comparisons. Antiproliferative activity was determined by
the inhibition of 3H-thymidine incorporation in HaCaT. c Results show percentages of inhibition (%I) at 5 µM. IC50 was determined for
those compounds that reached 50% inhibition. Statistical evaluation included one-way analysis of variance followed by Dunnett’s t-test
d
for multiple comparisons. PGE2 levels were determined by RIA. NS-398 was assayed as COX-2 reference inhibitor. Cytotoxicity was
determined by LDH release (mU) after 24 h treatment with 5 µM test compound. Data represent mean ( SEM (n ) 6-12). e P < 0.05.
f P < 0.01.
allyloxybenzoic acid, mp 167-169 °C (from ethanol), which was
converted to p-allyloxybenzoyl chloride with thionyl chloride
at reflux in dimethylformamide. The rest of the reagents used
in the biological tests were obtained from Sigma Chemicals
(St. Louis, MO).
Syn th esis of Diben zoyl Ava r ol (3), Di-p-m eth oxyben -
zoyl Ava r ol (4), a n d Di-p-a llyloxyben zoyl Ava r ol (5).
Benzoyl chloride, p-methoxybenzoyl chloride, and p-allyloxy-
benzoyl chloride (150 mg each) were individually added to a
solution of avarol (100 mg) in pyridine (2 mL) for 1 h at reflux.
The excess solvent reagents were removed in vacuo, and the
residues were partitioned between H2O and Et2O. The ether
extracts were dried over anhydrous Na2SO4, and the solvent
was evaporated to obtain an amorphous solid, which was
purified on a silica gel column (n-hexane-Et2O, 4:1). The
esters were crystallized from EtOH to give compounds 3-5
(yield 100% for each one).
Diben zoyl a va r ol (3): mp 158-159 °C; [R]25D +7.4° (c 0.13,
CHCl3); UV (MeOH) λmax (log ꢀ) 234 (4.13), 228 (4.11); 1H NMR
(CDCl3) δ 8.23 (4H, d, J ) 7.8 Hz, H2′′ and H6′′), 7.66 (2H, m,
H4′′), 7.53 (4H, m, H3′′ and H5′′), 7.14 (3H, m, H3′, H4′, and
H6′); EIMS m/z 522 [M]+ (2), 507 (1), 417 (5), 332 (30), 191
(100), 105 (95); HREIMS m/z 522.2773 (calcd for C35H38O4,
522.2770).
from respiratory burst of neutrophils infiltrated in the
psoriatic skin.13
In conclusion, the present study suggests that the
hydroquinone moiety and thiosalicylic function of com-
pound 6 can offer interesting anti-inflammatory properties
as antioxidant and inhibitor of PGE2 release. In addition,
the results obtained with compound 7 and its improved
ratio of antiproliferative activity to cytotoxicity as compared
to anthralin can be interesting as a possible antipsoriatic
drug. These results confirm that pharmacological study of
avarol and its derivatives can offer opportunities for
discovering novel therapeutic agents.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Melting points were
determined using a Kofler hot-stage microscope and are
uncorrected. Optical rotations were measured on a J ASCO DIP
370 polarimeter, using a 10 cm microcell. UV spectra were
1
obtained on a Varian DMS 90 spectrophotometer. H and 13C
NMR spectra were recorded at 500 and 125 MHz, respectively,
on a Bruker AMX-500 spectrometer in CDCl3, using the
residual CDCl3 resonance at 7.26 and 77.0 ppm as internal
references, respectively. Only chemical shifts of hydroquinone
and acyl residues are reported because all other signals
belonging to the sesquiterpenoid portion were earlier re-
ported.25,31 LRMS and HRMS were recorded on a J EOL J MS
D-300 and an AEI MS-50, respectively. Column chromatog-
raphy was carried out on Merck silica gel 60.
Ma ter ia ls. Avarol (1) was isolated from Dysidea avara,25
which was collected in the Bay of Naples, Italy. Benzoyl
chloride, p-methoxybenzoyl chloride, allyl bromide, p-hydroxy-
benzoic acid, thiosalicylic acid, methylamine, vanillylamine,
tryptamine, benzylamine, and p-methoxybenzylamine were
obtained from Sigma-Aldrich (Milano, Italy). p-Allyloxybenzoyl
chloride was obtained by adding allyl bromide to a solution of
p-hydroxybenzoic acid dissolved in 10% KOH-methanol,
maintaining the solution at reflux for 1 h. Workup with
aqueous 1 N HCl and extraction with Et2O afforded p-
Di-p-m eth oxyben zoyl a va r ol (4): mp 175-177 °C; [R]25
D
+10.2° (c 0.11, CHCl3); UV (MeOH) λmax (logꢀ) 262 (4.29), 219
1
(4.07); H NMR (CDCl3) δ 8.16 (4H, d, J ) 8.8 Hz, H2′′ and
H6′′), 7.11 (3H, m, H3′, H4′, and H6′), 6.99 (4H, d, J ) 8.8 Hz,
H3′′ and H5′′); EIMS m/z 582 [M]+ (0.1), 447 (3), 392 (15), 191
(90), 135 (100); HREIMS m/z 582.2977 (calcd for C37H42O6,
582.2981).
Di-p-a llyloxyben zoyl a va r ol (5): mp 149-150 °C; [R]25
D
+10.1° (c 0.11, CHCl3); UV (MeOH) λmax (log ꢀ) 265 (4.77), 215
1
(4.25); H NMR (CDCl3) δ 8.15 (4H, d, J ) 8.8 Hz, H2′′ and
H6′′), 7.10 (3H, m, H3′, H4′, and H6′), 7.00 (4H, d, J ) 8.8 Hz,
H3′′ and H5′′), 6.08 (2H, m, H2′′′), 5.45 (2H, d, J ) 17.3 Hz,
H3a′′′), 5.34 (2H, d, J ) 10.5 Hz, H3b′′′), 4.64 (4H, d, J ) 5.2
Hz, H1′′′); EIMS m/z 634 [M]+ (0.3), 594 (0.3), 473 (1), 444 (15),
191 (90), 161 (100); HREIMS m/z 634.3300 (calcd for C41H46O6,
634.3294).

1462 J ournal of Natural Products, 2004, Vol. 67, No. 9
Amigo´ et al.
Syn th esis of Ava r ol-3′-th iosa licyla te (6). Thiosalicylic
acid (100 mg) dissolved in EtOH (10 mL) was added to a
solution of avarone (2) (100 mg) in EtOH (20 mL) and stirred
for 5 min at room temperature. After evaporation of EtOH,
the residue was chromatographed on a Si gel column and
eluted with petroleum ether-Et2O-HOAc (7:3:0.1) to give
avarol-3′-thiosalicylate (6) (90 mg): mp 113-115 °C (CHCl3-
graphed on a Si gel column and eluted with petroleum ether-
Et2O (8:2) to give 3′-p-methoxybenzylaminoavarone (15) as the
more polar component (28 mg): mp 90-91 °C (hexane); [R]25
D
-58.2° (c 0.009, CHCl3); UV (MeOH) λmax (log ꢀ) 283 (3.79),
485 (3.38); 1H NMR (CDCl3) δ 6.37 (H-6′, d, J ) 2.3 Hz), 5.48
(H-4′, d, J ) 2.3 Hz); EIMS m/z 447 [M]+ (10), 432 (30), 257
(95), 173 (75), 160 (30), 138 (80), 121 (100); HREIMS m/z
447.2770 (calcd for C29H37NO3, 447.2773), and 4′-p-methoxy-
benzylaminoavarone (16) as the less polar component (38
MeOH); [R]25 -0.33° (c 0.014, CHCl3); UV (MeOH) λmax (log
D
ꢀ) 317 (4.07); 1H NMR (CDCl3) δ 6.91 (H-6′, d, J ) 2.5 Hz),
6.79 (H-4′, d, J ) 2.5 Hz); EIMS m/z 468 [M + 2]+ (0.4), 466
[M]+ (10), 276 (18), 258 (22), 189 (30), 135 (30), 107 (35), 95
(100); HREIMS m/z 466.2183 (calcd for C28H34O4S, 466.2178).
Syn th esis of 7 a n d 8. Avarone (2) (200 mg) was treated
with MeNH2-HCl as previously described,9 and 42 mg of 7
and 73 mg of 8 were recovered. Compounds 7 and 8 were
identified by comparison with authentic samples.
mg): mp 131-132 °C (hexane); [R]25 -80.1° (c 0.01, CHCl3);
D
UV (MeOH) λmax (log ꢀ) 284 (4.10), 486 (3.43); 1H NMR (CDCl3)
δ 6.37 (H-6′, s), 5.51 (H-3′, s); EIMS m/z 447 [M]+ (10), 432
(15), 257 (100), 175 (55), 160 (80), 138 (70), 121 (95); HREIMS
m/z 447.2777 (calcd for C29H37NO3, 447.2773).
Biologica l Test s. F r ee-R a d ica l Sca ven gin g Act ivit y.
TLC Au togr a p h ic Assa y. A 5 µg sample of compounds 1 and
3-16 was applied on a TLC plate. After developing [eluent:
light petroleum-Et2O (1:1)] and drying, TLC plates were
sprayed with a 0.2% DPPH solution in MeOH. The plates were
examined 30 min after spraying. Active compounds (1, 6, 9,
and 10) appear as yellow spots against a purple background.
Assa y in Solu tion . Solutions of compounds 1, 6, 9, and 10
in MeOH, at different concentrations, were prepared and
adjusted to 2 mL total volume with 0.7 mL of DPPH-MeOH
solution (6 mg/50 mL; 0.1 mM final concentration). The
absorbance at 517 nm was determined after 30 min, and the
percent free radical inhibition was calculated and plotted to
obtain the IC50 value. The IC50 value denotes the concentration
of compound required to scavenge 50% DPPH free radical.
ROS Gen er a tion in Hu m a n Neu tr op h ils. Human neu-
trophils were obtained from citrated blood of healthy volun-
teers and purified as previously described.32 Neutrophils (2.5
× 106 cells/mL) were incubated with luminol (40 µM) and
stimulated with 1 µM 12-O-tetradecanoyl phorbol 13-acetate
(TPA) for 7 min. ROS generation was determined as chemi-
luminescence recorded in a Microbeta trilux counter (Wallac,
Turku, Finland).
Cytotoxicity Assa y. The cytotoxicity of products was
assessed by determination of lactate dehydrogenase (LDH)
release in supernatants obtained after treatment of neutro-
phils and keratinocytes with tested compounds.28
An tip r olifer a tive Assa y. The human keratinocyte cell line
HaCaT was provided by Dr. N. E. Fusenig (Heidelberg,
Germany). The cell line was cultured in DMEM medium with
10% fetal calf serum (FCS), 100 U/mL penicillin, and 100 µg/
mL streptomycin, in a humidified incubator (5% CO2 at 37 °C.)
Keratinocytes (4 × 104/200 µL/well) were grown during 24 h
on 96-well multidishes. The medium was replaced, and test
compounds or vehicle (1% EtOH) was added. After 24 h of
incubation, cells were pulsed for 6 h with methyl-3[H]-
thymidine (1 µCi/mL). Cells were washed and thymidine
incorporation was measured with a Microbeta trilux counter
after addition of 200 µL of liquid scintillation counting. All the
compounds were tested for antiproliferative activity in com-
parison to the antipsoriatic drug anthralin.
P GE2 Gen er a tion Assa y. HaCaT cells (1 × 104/200µL/well)
were seeded in 96-well plates and cultivated for 24 h. After
being washed, test compounds (5 µM) and arachidonic acid (10
µM) were added for 24 h incubation. Finally, supernatants
were collected for the determination of PGE2 by radioimmu-
noassay.33
Syn th esis of 9 a n d 10. Vanillylamine (200 mg) was
dissolved in a saturated solution of NaHCO3 (30 mL), added
to a solution of avarone (2) (100 mg) in EtOH (30 mL), and
stirred for 24 h at room temperature. After evaporation of
EtOH, the remaining aqueous solution was extracted with
CHCl3, and the extract was chromatographed on a Si gel
column and eluted with petroleum ether-Et2O (7:3). The more
polar component was 3′-vanillylaminoavarone (9) (30 mg): mp
145-146 °C (CHCl3-MeOH); [R]25D -72.4° (c 0.01, CHCl3); UV
1
(MeOH) λmax (log ꢀ) 283 (3.82), 486 (3.18); H NMR (CDCl3) δ
6.38 (H-6′, d, J ) 2.3 Hz), 5.50 (H-4′, d, J ) 2.3 Hz); EIMS
m/z 463 [M]+ (1.5), 448 (3), 312 (2), 273 (30), 191 (20), 175 (20),
139 (90) 137 (100); HREIMS m/z 463.2725 (calcd for C29H37
-
NO4, 463.2722). The less polar component was 4′-vanillylami-
noavarone (10) (36 mg): mp 159-161 °C (CHCl3-MeOH);
[R]25D -50.4° (c 0.01, CHCl3); UV (MeOH) λmax (log ꢀ) 284 (4.14),
486 (3.33); 1H NMR (CDCl3) δ 6.38 (H-6′, s), 5.52 (H-3′, s);
EIMS m/z 463 [M]+ (5), 448 (6), 312 (18), 274 (90), 273 (85)
189 (60), 175 (50), 139 (90) 137 (100); HREIMS m/z 463.2719
(calcd for C29H37NO4, 463.2722).
Syn th esis of 11 a n d 12. Tryptamine (200 mg) dissolved
in a saturated solution of NaHCO3 (30 mL) was added to a
solution of avarone (2) (100 mg) in EtOH (30 mL). After the
usual workup, the CHCl3 extract was chromatographed on a
Si gel column and eluted with petroleum ether-Et2O (6:4) to
give 3′-tryptaminoavarone (11) as the more polar component
(32 mg): mp 183-184 °C (CHCl3-MeOH); [R]25 -55.0° (c
D
0.015, CHCl3); UV (MeOH) λmax (log ꢀ) 283 (4.17), 493 (3.40);
1H NMR (CDCl3) δ 6.26 (H-6′, d, J ) 2.3 Hz), 5.37 (H-4′, d, J
) 2.3 Hz); EIMS m/z 470 [M]+ (1.5), 455 (2), 341 (8), 282 (12),
151 (65), 130 (100); HREIMS m/z 470.2937 (calcd for C31H38
-
N2O2, 470.2933), and 4′-tryptaminoavarone (12) as the less
polar component (18 mg): mp 163-164 °C (CHCl3-MeOH);
[R]25D -33.5° (c 0.01, CHCl3); UV (MeOH) λmax (log ꢀ) 282 (4.41),
496 (3.84); 1H NMR (CDCl3) δ 6.23 (H-6′, s), 5.38 (H-3′, s);
EIMS m/z 470 [M]+ (8), 455 (12), 340 (18), 281 (90), 189 (10),
175 (50), 150 (90) 144 (900), 130 (100); HREIMS m/z 470.2935
(calcd for C31H38N2O2, 470.2933).
Syn th esis of 13 a n d 14. Benzylamine (2 mL) dissolved in
a saturated solution of NaHCO3 (30 mL) was added to a
solution of avarone (2) (100 mg) in EtOH (30 mL). After the
usual workup, the CHCl3 extract was chromatographed on a
Si gel column and eluted with petroleum ether-Et2O (8:2) to
give 3′-benzylaminoavarone (13) as the more polar component
(33 mg): mp 82-83 °C (hexane); [R]25D -90.3° (c 0.015, CHCl3);
UV (MeOH) λmax (log ꢀ) 288 (3.82), 486 (3.44); 1H NMR (CDCl3)
δ 6.38 (H-6′, d, J ) 2.3 Hz), 5.48 (H-4′, d, J ) 2.3 Hz); EIMS
m/z 417 [M]+ (3), 402 (30), 227 (100), 173 (75), 150 (80), 138
(95); HREIMS m/z 417.2663 (calcd for C28H35NO2, 417.2668),
and 4′-benzylaminoavarone (14) as the less polar component
Sta tistica l An a lysis. The results are presented as mean
( SEM. Inhibitory concentration 50% (IC50) values were
calculated from at least four significant concentrations (n )
6). The level of statistical significance was determined by
analysis of variance (ANOVA), followed by Dunnett’s t-test for
multiple comparisons.
(46 mg): mp 107-108 °C (hexane); [R]25 -44.0° (c 0.02,
D
CHCl3); UV (MeOH) λmax (log ꢀ) 290 (4.10), 482 (3.51); 1H NMR
(CDCl3) δ 6.38 (H-6′, s), 5.48 (H-3′, s); EIMS m/z 417 [M]+ (5),
402 (16), 227 (100), 175 (20), 149 (15), 138 (10); HREIMS m/z
417.2670 (calcd for C28H35NO2, 417.2668).
Ack n ow led gm en t. This work was supported by grant SAF
2001-2639 from CICYT Spain and by the European Commis-
sion (Project Sponge, contract number QLK3-1999-00672. M.A.
was the recipient of a scholarship from Spanish Ministerio de
Educacio´n Cultura y Deporte.
Syn th esis of 15 a n d 16. 4-Methoxybenzylamine (1.8 mL)
dissolved in a saturated solution of NaHCO3 (30 mL) was
added to a solution of avarone (2) (100 mg) in EtOH (30 mL).
After the usual workup, the CHCl3 extract was chromato-

Potential Antipsoriatic Avarol Derivatives
J ournal of Natural Products, 2004, Vol. 67, No. 9 1463
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